Differentiated adaptative genetic architecture and language-related demographical history in South China inferred from 619 genomes from 56 populations.

BACKGROUND: The underrepresentation of human genomic resources from Southern Chinese populations limited their health equality in the precision medicine era and complete understanding of their genetic formation, admixture, and adaptive features. Besides, linguistical and genetic evidence supported the controversial hypothesis of their origin processes. One hotspot case was from the Chinese Guangxi Pinghua Han people (GPH), whose language was significantly similar to Southern Chinese dialects but whose uniparental gene pool was phylogenetically associated with the indigenous Tai-Kadai (TK) people. Here, we analyzed genome-wide SNP data in 619 people from four language families and 56 geographically different populations, in which 261 people from 21 geographically distinct populations were first reported here. RESULTS: We identified significant population stratification among ethnolinguistically diverse Guangxi populations, suggesting their differentiated genetic origin and admixture processes. GPH shared more alleles related to Zhuang than Southern Han Chinese but received more northern ancestry relative to Zhuang. Admixture models and estimates of genetic distances showed that GPH had a close genetic relationship with geographically close TK compared to Northern Han Chinese, supporting their admixture origin hypothesis. Further admixture time and demographic history reconstruction supported GPH was formed via admixture between Northern Han Chinese and Southern TK people. We identified robust signatures associated with lipid metabolisms, such as fatty acid desaturases (FADS) and medically relevant loci associated with Mendelian disorder (GJB2) and complex diseases. We also explored the shared and unique selection signatures of ethnically different but linguistically related Guangxi lineages and found some shared signals related to immune and malaria resistance. CONCLUSIONS: Our genetic analysis illuminated the language-related fine-scale genetic structure and provided robust genetic evidence to support the admixture hypothesis that can explain the pattern of observed genetic diversity and formation of GPH. This work presented one comprehensive analysis focused on the population history and demographical adaptative process, which provided genetic evidence for personal health management and disease risk prediction models from Guangxi people. Further large-scale whole-genome sequencing projects would provide the entire landscape of southern Chinese genomic diversity and their contributions to human health and disease traits.

GMP-grade human neural progenitors delivered subretinally protect vision in rat model of retinal degeneration and survive in minipigs.

BACKGROUND: Stem cell products are increasingly entering early stage clinical trials for treating retinal degeneration. The field is learning from experience about comparability of cells proposed for preclinical and clinical use. Without this, preclinical data supporting translation to a clinical study might not adequately reflect the performance of subsequent clinical-grade cells in patients. METHODS: Research-grade human neural progenitor cells (hNPC) and clinical-grade hNPC (termed CNS10-NPC) were injected into the subretinal space of the Royal College of Surgeons (RCS) rat, a rodent model of retinal degeneration such as retinitis pigmentosa. An investigational new drug (IND)-enabling study with CNS10-NPC was performed in the same rodent model. Finally, surgical methodology for subretinal cell delivery in the clinic was optimized in a large animal model with Yucatan minipigs. RESULTS: Both research-grade hNPC and clinical-grade hNPC can survive and provide functional and morphological protection in a dose-dependent fashion in RCS rats and the optimal cell dose was defined and used in IND-enabling studies. Grafted CNS10-NPC migrated from the injection site without differentiation into retinal cell phenotypes. Additionally, CNS10-NPC showed long-term survival, safety and efficacy in a good laboratory practice (GLP) toxicity and tumorigenicity study, with no observed cell overgrowth even at the maximum deliverable dose. Finally, using a large animal model with the Yucatan minipig, which has an eye size comparable to the human, we optimized the surgical methodology for subretinal cell delivery in the clinic. CONCLUSIONS: These extensive studies supported an approved IND and the translation of CNS10-NPC to an ongoing Phase 1/2a clinical trial (NCT04284293) for the treatment of retinitis pigmentosa.

Aquatic Bacteria Rheinheimera tangshanensis New Ability for Mercury Pollution Removal.

To explore the strong tolerance of bacteria to Hg pollution, aquatic Rheinheimera tangshanensis (RTS-4) was separated from industrial sewage, with a maximum Hg(II) tolerant concentration of 120 mg/L and a maximum Hg(II) removal rate of 86.72 ± 2.11%, in 48 h under optimum culture conditions. The Hg(II) bioremediation mechanisms of RTS-4 bacteria are as follows: (1) the reduction of Hg(II) through Hg reductase encoded by the mer operon; (2) the adsorption of Hg(II) through the production of extracellular polymeric substances (EPSs); and (3) the adsorption of Hg(II) using dead bacterial biomass (DBB). At low concentrations [Hg(II) ≤ 10 mg/L], RTS-4 bacteria employed Hg(II) reduction and DBB adsorption to remove Hg(II), and the removal percentages were 54.57 ± 0.36% and 45.43 ± 0.19% of the total removal efficiency, respectively. At moderate concentrations [10 mg/L < Hg(II) ≤ 50 mg/L], all three mechanisms listed above coexisted, with the percentages being 0.26 ± 0.01%, 81.70 ± 2.31%, and 18.04 ± 0.62% of the total removal rate, respectively. At high concentrations [Hg(II) > 50 mg/L], the bacteria primary employed EPS and DBB adsorption to remove Hg(II), where the percentages were 19.09 ± 0.04% and 80.91 ± 2.41% of the total removal rate, respectively. When all three mechanisms coexisted, the reduction of Hg(II) occurred within 8 h, the adsorption of Hg(II) by EPSs and DBB occurred within 8-20 h and after 20 h, respectively. This study provides an efficient and unused bacterium for the biological treatment of Hg pollution.

First Report of Rhizoctonia solani AG-2-2 IIIB Causing Foliar Blight on Bletilla striata in China.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae), a perennial plant, is a traditional Chinese herb (known as baiji) used to treat hemorrhage, scalding injuries, gastric ulcers, pulmonary diseases, and inflammation (Zu et al. 2019). In May 2019, foliar blight symptoms were observed on approximately 25% of B. striata (cv. Guiji No.1) plants in three plantations (∼4.5 hectares in total) in Ziyuan County, Guangxi Province, China. Initial symptoms were light brown, irregular, water-soaked spots on the plant leaves. Several spots often merged, forming large, irregular, lesions that extended onto the stem after a week and led to leaf abscission, and even plant death. To determine the causal agent, 5-mm squares cut from the margin of 6 infected leaves were surface disinfected in 1% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 28°C (12-h light-dark cycle) for 3 days. The emerging hyphal tip of a single mycelium was transferred to PDA to obtain pure cultures of the isolates. Twenty isolates were obtained, and 10 isolates (50%) were initially white before turning light brown (∼4 days). Septate hyphae were 4.29 to 10.75 µm (average 6.42 µm) in diameter and branched at right angles with a constriction at the origin of the branch point. Staining with 1% safranin O and 3% KOH solution (Bandoni 1979) revealed multinucleated cells (3 to 9 nuclei per cell, n = 142). This morphology was typical of Rhizoctonia solani Kühn (Meyer et al. 1990). For species confirmation by molecular identification, three isolates (BJ101.6, BJ101.11, and BJ102.2) were cultured on PDA for 4 days, then DNA was extracted from the mycelium using the CTAB method (Guo et al. 2000), and the ribosomal ITS1-5.8S-ITS2 region was amplified by PCR using the universal fungal primers ITS1 and ITS4 (White et al. 1990). Internal transcribed spacer (ITS) sequences of strains BJ101.6, BJ101.11, and BJ102 (deposited in GenBank under accession nos MT406271, MT892815, and MT892814, respectively) had over 99% similarity with those of R. solani AG-2-2 IIIB in GenBank (accession nos JX913810 and AB054858) (Carling et al. 2002; Hong et al. 2012). Phylogenetic analysis using ITS sequences showed that the isolates clustered monophyletically with strains of R. solani AG-2-2 IIIB. The AG of the isolates was confirmed by their ability to grow well on PDA at 35°C, which separates AG-2-2 IIIB from AG-2-2 IV (Inokuti et al. 2019). Based on morphological characteristics and nucleotide sequence analysis, the isolates were identified as R. solani AG-2-2 IIIB. Pathogenicity was tested using 1.5-year-old B. striata (cv. Guiji No.1) plants grown in a perlite and peat moss mixture (1:3) in 7-cm pots. Healthy leaves on plants were inoculated with an aqueous suspension (approximately 1 × 105 hyphal fragments/mL, 100 µL) prepared from cultures of strains BJ101.6, BJ101.11, and BJ102.2, each isolate was inoculated onto three plants; three other plants with sterile water served as controls. All plants were enclosed in transparent plastic bags and incubated in a greenhouse at 28°C for 14 days (12-h photoperiod). Three days post-inoculation, leaves exposed to the mycelial fragments had symptoms similar to those originally observed in the field. No symptoms were detected on control plants. Experiments were replicated three times with similar results. To fulfill Koch's postulates, R. solani AG-2-2 IIIB was re-isolated on PDA from symptomatic leaves and confirmed by sequencing, whereas no fungus was isolated from the control plants. To our knowledge, this is the first report of R. solani AG-2-2 IIIB causing foliar blight on B. striata in China, and these findings will be useful for further control strategies and research.

First Report of Anthracnose on Bletilla striata Caused by Colletotrichum fructicola in China.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is a traditional Chinese medicinal plant. In April 2018 and 2019, a leaf spot disease was observed on ∼20% of B. striata plants in two fields (∼1.4 h) in Guilin, Guangxi Province, China (Fig.1 A). Small, circular, brown spots were initially observed on the leaf surfaces, which progressively expanded into large, sunken, dark brown, necrotic areas. As the disease progressed, lesions merged into large, irregular spots, ultimately resulting in abscission. To determine the causal agent, small pieces (5 mm x 5 mm) were collected from the infected leaf tissues (n = 18), surface sterilized in 1% NaOCl for 2 min, and rinsed three times with sterile water. Then, the tissues were placed on potato dextrose agar (PDA) with chloramphenicol (0.1 g/L) and incubated under 12 h photoperiod at 26°C for 3 days. Seventeen isolates were obtained, of which twelve isolates with similar morphological characteristics were obtained from the germinated spores on PDA. Seven-day-old colonies on PDA appeared cottony, pale white to pale gray from above, and grayish-green from below. Conidia of strain BJ-101.3 were hyaline, aseptate, straight, and cylindrical, with rounded ends (Fig.1 E-G), measuring 11.3 to 15.9 µm × 4.0 to 6.4 µm (n = 50). Appressoria were brown to dark brown, with different shapes and a smooth edge (Fig.1 H-I), measuring 6.3 to 10.0 µm × 4.1 to 8.0 µm (n = 50). Morphological features were similar to C. gloeosporioides species complex (Weir et al. 2012, Fuentes-Aragón et al. 2018). For molecular identification, DNA was extracted from two isolates BJ-101.3 and BJ-101.13, following the CTAB method (Guo et al. 2000). The internal transcribed spacer (ITS) region, partial actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), manganese superoxide dismutase (SOD2), beta-tubulin (TUB2), glutamine synthetase (GS), and Apn2-Mat1-2 intergenic spacer and partial mating-type (ApMat) genes were amplified by PCR and sequenced (Weir et al. 2012, Silva et al. 2012, Vieira et al. 2017). The obtained sequences were deposited in GenBank (MW386818, MW386819, MW403508 to MW403519, and MW888410 to MW888413). BLASTN analysis of the obtained sequences showed 99% identity with those of C. fructicola (JX010165，JX010033, FJ917508, FJ907426, JX009866, JX010095, JX010327, JX010405, JQ807838) (Weir et al. 2012, Liu et al. 2015). A phylogenetic tree based on the concatenated sequences confirmed the isolates as C. fructicola (Fig.2). Furthermore, pathogenicity tests were conducted on six 1.5-year-old B. striata plants. Healthy leaves on the plants were inoculated with the conidial suspensions (106 conidia/mL; 10 µL) of the strains BJ-101.3 and BJ101.13. The conidial suspension of each isolate was inoculated onto at least three leaves. Another three plants inoculated with sterile water served as the control. All plants were covered with transparent plastic bags and incubated in a greenhouse at 26°C for 14 days with a 12 h photoperiod. Nine days post-inoculation, the inoculated leaves showed leaf spot symptoms, while the control plants remained symptomless (Fig.1 B-C). The experiments repeated three times showed similar results. Finally, C. fructicola was consistently reisolated from the infected leaves and confirmed by morphology and sequencing, fulfilling Koch's postulates. The outcome of this study will help in developing effective management measures against anthracnose of B. striata.

Identification of Fusarium ipomoeae as the causative agent of leaf spot disease in Bletilla striata in China.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is traditionally used for hemostasis and detumescence in China. In April 2019, a leaf spot disease on B. striata was observed in plant nurseries in Guilin, Guangxi Province, China, with an estimated incidence of ~30%. Initial symptoms include the appearance of circular or irregular brown spots on leaf surfaces, which progressively expand into large, dark brown, necrotic areas. As lesions coalesce, large areas of the leaf die, ultimately resulting in abscission. To isolate the pathogen, representative samples exhibiting symptoms were collected, leaf tissues (5 × 5 mm) were cut from the junction of diseased and healthy tissue, surface-disinfected in 1% sodium hypochlorite solution for 2 min, rinsed three times in sterile water, plated on potato dextrose agar (PDA) medium, and incubated at 28°C (12-h light-dark cycle) for 3 days. Hyphal tips from recently germinated spores were transferred to PDA to obtain pure cultures. Nine fungal isolates with similar morphological characteristics were obtained. Colonies on PDA were villose, had a dense growth of aerial mycelia and appeared pinkish white from above and greyish orange at the center and pinkish-white at the margin on the underside. Macroconidia were smooth, and hyaline, with a dorsiventral curvature, hooked to tapering apical cells, and 3- to 5-septate. Three-septate macroconidia were 21.2 to 32.1 × 2.4 to 3.9 µm (mean ± SD: 26.9 ± 2.5 × 3.2 ± 0.4 µm, n = 30); 4-septate macroconidia were 29.5 to 38.9 × 3.0 to 4.3 µm (mean ± SD: 33.5 ± 2.6 × 3.6 ± 0.3 µm, n = 40); and 5-septate macroconidia were 39.3 to 55.6 × 4.0 to 5.4 µm (mean ± SD: 48.0 ± 3.9 × 4.5 ± 0.3 µm, n = 50). These morphological characteristics were consistent with F. ipomoeae, a member of the Fusarium incarnatum-equiseti species complex (FIESC) (Wang et al. 2019). To confirm the fungal isolate's identification, the genomic DNA of the single-spore isolate BJ-22.3 was extracted using the CTAB method (Guo et al. 2000). The internal transcribed space (ITS) region of rDNA, translation elongation factor-1 alpha (TEF-1α), and partial RNA polymerase second largest subunit (RPB2) were amplified using primer pairs [ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (O'Donnell et al. 1998), and 5f2/11ar (Liu, Whelen et al. 1999, Reeb, Lutzoni et al. 2004), respectively]. The ITS (MT939248), TEF-1α (MT946880), and RPB2 (MT946881) sequences of the BJ-22.3 isolate were deposited in GenBank. BLASTN analysis of these sequences showed over 99% nucleotide sequence identity with members of the FIESC: the ITS sequence showed 99.6% identity (544/546 bp) to F. lacertarum strain NRRL 20423 (GQ505682); the TEF-1α sequence showed 99.4% similarity (673/677 bp) to F. ipomoeae strain NRRL 43637 (GQ505664); and the RPB2 sequence showed 99.6% identity (1883/1901 bp) to F. equiseti strain GZUA.1657 (MG839492). Phylogenetic analysis using concatenated sequences of ITS, TEF-1α, and RPB2 showed that BJ-22.3 clustered monophyletically with strains of F. ipomoeae. Therefore, based on morphological and molecular characteristics, the isolate BJ-22.3 was identified as F. ipomoeae. To verify the F. ipomoeae isolate's pathogenicity, nine 1.5-year-old B. striata plants were inoculated with three 5 × 5 mm mycelial discs of strain BJ-22.3 from 4-day-old PDA cultures. Additionally, three control plants were inoculated with sterile PDA discs. The experiments were replicated three times. All plants were enclosed in transparent plastic bags and incubated in a greenhouse at 26°C for 14 days. Four days post-inoculation, leaf spot symptoms appeared on the inoculated leaves, while no symptoms were observed in control plants. Finally, F. ipomoeae was consistently re-isolated from leaf lesions from the infected plants. To our knowledge, this is the first report of F. ipomoeae causing leaf spot disease on B. striata in China. The spread of this disease might pose a serious threat to the production of B. striata. Growers should implement disease management to minimize the risks posed by this pathogen.

In Vitro and In Vivo Proteomic Comparison of Human Neural Progenitor Cell-Induced Photoreceptor Survival.

Retinal degenerative diseases lead to blindness with few treatments. Various cell-based therapies are aimed to slow the progression of vision loss by preserving light-sensing photoreceptor cells. A subretinal injection of human neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) rat model of retinal degeneration has aided in photoreceptor survival, though the mechanisms are mainly unknown. Identifying the retinal proteomic changes that occur following hNPC treatment leads to better understanding of neuroprotection. To mimic the retinal environment following hNPC injection, a co-culture system of retinas and hNPCs is developed. Less cell death occurs in RCS retinal tissue co-cultured with hNPCs than in retinas cultured alone, suggesting that hNPCs provide retinal protection in vitro. Comparison of ex vivo and in vivo retinas identifies nuclear factor (erythroid-derived 2)-like 2 (NRF2) mediated oxidative response signaling as an hNPC-induced pathway. This is the first study to compare proteomic changes following treatment with hNPCs in both an ex vivo and in vivo environment, further allowing the use of ex vivo modeling for mechanisms of retinal preservation. Elucidation of the protein changes in the retina following hNPC treatment may lead to the discovery of mechanisms of photoreceptor survival and its therapeutic for clinical applications.

Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity.

Human umbilical tissue-derived cells (hUTC or palucorcel) are currently under clinical investigation for the treatment of geographic atrophy, a late stage of macular degeneration, but how hUTC transplantation mediates vision recovery is not fully elucidated. Subretinal administration of hUTC preserves visual function in the Royal College of Surgeons (RCS) rat, a genetic model of retinal degeneration caused by Mertk loss of function. hUTC secrete synaptogenic and neurotrophic factors that improve the health and connectivity of the neural retina. Therefore, we investigated the progression of synapse and photoreceptor loss and whether hUTC treatment preserves photoreceptors and synaptic connectivity in the RCS rats of both sexes. We found that RCS retinas display significant deficits in synaptic development already by postnatal day 21 (P21), before the onset of photoreceptor degeneration. Subretinal transplantation of hUTC at P21 is necessary to rescue visual function in RCS rats, and the therapeutic effect is enhanced with repeated injections. Synaptic development defects occurred concurrently with morphological changes in Müller glia, the major perisynaptic glia in the retina. hUTC transplantation strongly diminished Müller glia reactivity and specifically protected the α2δ-1-containing retinal synapses, which are responsive to thrombospondin family synaptogenic proteins secreted by Müller glia. Müller glial reactivity and reduced synaptogenesis observed in RCS retinas could be recapitulated by CRISPR/Cas9-mediated loss-of-Mertk in Müller glia in wild-type rats. Together, our results show that hUTC transplantation supports the health of retina at least in part by preserving the functions of Müller glial cells, revealing a previously unknown aspect of hUTC transplantation-based therapy.SIGNIFICANCE STATEMENT Despite the promising effects observed in clinical trials and preclinical studies, how subretinal human umbilical tissue-derived cell (hUTC) transplantation mediates vision improvements is not fully known. Using a rat model of retinal degeneration, the RCS rat (lacking Mertk), here we provide evidence that hUTC transplantation protects visual function and health by protecting photoreceptors and preserving retinal synaptic connectivity. Furthermore, we find that loss of Mertk function only in Müller glia is sufficient to impair synaptic development and cause activation of Müller glia. hUTC transplantation strongly attenuates the reactivity of Müller glia in RCS rats. These findings highlight novel cellular and molecular mechanisms within the neural retina, which underlie disease mechanisms and pinpoint Müller glia as a novel cellular target for hUTC transplantation.

A two-step mutation process in the double WS1 homologs drives the evolution of burley tobacco, a special chlorophyll-deficient mutant with abnormal chloroplast development.

MAIN CONCLUSION: The functional homologs WS1A and WS1B, identified by map-based cloning, control the burley character by affecting chloroplast development in tobacco, contributing to gene isolation and genetic improvement in polyploid crops. Burley represents a special type of tobacco (Nicotiana tabacum L.) cultivar that is characterized by a white stem with a high degree of chlorophyll deficiency. Although important progress in the research of burley tobacco has been made, the molecular mechanisms underlying this character remain unclear. Here, on the basis of our previous genetic analyses and preliminary mapping results, we isolated the White Stem 1A (WS1A) and WS1B genes using a map-based cloning approach. WS1A and WS1B are functional homologs with completely identical biological functions and highly similar expression patterns that control the burley character in tobacco. WS1A and WS1B are derived from Nicotiana sylvestris and Nicotiana tomentosiformis, the diploid ancestors of Nicotiana tabacum, respectively. The two genes encode zinc metalloproteases of the M50 family that are highly homologous to the Ethylene-dependent Gravitropism-deficient and Yellow-green 1 (EGY1) protein of Arabidopsis and the Lutescent 2 (L2) protein of tomato. Transmission electron microscopic examinations indicated that WS1A and WS1B are involved in the development of chloroplasts by controlling the formation of thylakoid membranes, very similar to that observed for EGY1 and L2. The genotyping of historical tobacco varieties revealed that a two-step mutation process occurred in WS1A and WS1B during the evolution of burley tobacco. We also discussed the strategy for gene map-based cloning in polyploid plants with complex genomes. This study will facilitate the identification of agronomically important genes in tobacco and other polyploid crops and provide insights into crop improvement via molecular approaches.

Morphological phenotyping and genetic analyses of a new chemical-mutagenized population of tobacco (Nicotiana tabacum L.).

MAIN CONCLUSION: A novel tobacco mutant library was constructed, screened, and characterized as a crucial genetic resource for functional genomics and applied research. A comprehensive mutant library is a fundamental resource for investigating gene functions, especially after the completion of genome sequencing. A new tobacco mutant population induced by ethyl methane sulfonate mutagenesis was developed for functional genomics applications. We isolated 1607 mutant lines and 8610 mutant plants with altered morphological phenotypes from 5513 independent M2 families that consisted of 69,531 M2 plants. The 2196 mutations of abnormal phenotypes in the M2 putative mutants were classified into four groups with 17 major categories and 51 subcategories. More than 60% of the abnormal phenotypes observed fell within the five major categories including plant height, leaf shape, leaf surface, leaf color, and flowering time. The 465 M2 mutants exhibited multiple phenotypes, and 1054 of the 2196 mutations were pleiotropic. Verification of the phenotypes in advanced generations indicated that 70.63% of the M3 lines, 84.87% of the M4 lines, and 95.75% of the M5 lines could transmit original mutant phenotypes of the corresponding M2, M3, and M4 mutant plants. Along with the increased generation of mutants, the ratios of lines inheriting OMPs increased and lines with emerging novel mutant phenotypes decreased. Genetic analyses of 18 stably heritable mutants showed that two mutants were double recessive, five were monogenic recessive, eight presented monogenic dominant inheritance, and three presented semi-dominant inheritance. The pleiotropy pattern, saturability evaluation, research prospects of genome, and phenome of the mutant populations were also discussed. Simultaneously, this novel mutant library provided a fundamental resource for investigating gene functions in tobacco.

In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa.

Reliable genome editing via Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 may provide a means to correct inherited diseases in patients. As proof of principle, we show that CRISPR/Cas9 can be used in vivo to selectively ablate the rhodopsin gene carrying the dominant S334ter mutation (Rho(S334)) in rats that model severe autosomal dominant retinitis pigmentosa. A single subretinal injection of guide RNA/Cas9 plasmid in combination with electroporation generated allele-specific disruption of Rho(S334), which prevented retinal degeneration and improved visual function.

Gene expression changes in the retina following subretinal injection of human neural progenitor cells into a rodent model for retinal degeneration.

PURPOSE: Retinal degenerative diseases (RDDs) affect millions of people and are the leading cause of vision loss. Although treatment options for RDDs are limited, stem and progenitor cell-based therapies have great potential to halt or slow the progression of vision loss. Our previous studies have shown that a single subretinal injection of human forebrain derived neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) retinal degenerate rat offers long-term preservation of photoreceptors and visual function. Furthermore, neural progenitor cells are currently in clinical trials for treating age-related macular degeneration; however, the molecular mechanisms of stem cell-based therapies are largely unknown. This is the first study to analyze gene expression changes in the retina of RCS rats following subretinal injection of hNPCs using high-throughput sequencing. METHODS: RNA-seq data of retinas from RCS rats injected with hNPCs (RCS(hNPCs)) were compared to sham surgery in RCS (RCS(sham)) and wild-type Long Evans (LE(sham)) rats. Differential gene expression patterns were determined with in silico analysis and confirmed with qRT-PCR. Function, biologic, cellular component, and pathway analyses were performed on differentially expressed genes and investigated with immunofluorescent staining experiments. RESULTS: Analysis of the gene expression data sets identified 1,215 genes that were differentially expressed between RCS(sham) and LE(sham) samples. Additionally, 283 genes were differentially expressed between the RCS(hNPCs) and RCS(sham) samples. Comparison of these two gene sets identified 68 genes with inverse expression (termed rescue genes), including Pdc, Rp1, and Cdc42ep5. Functional, biologic, and cellular component analyses indicate that the immune response is enhanced in RCS(sham). Pathway analysis of the differential expression gene sets identified three affected pathways in RCS(hNPCs), which all play roles in phagocytosis signaling. Immunofluorescent staining detected the increased presence of macrophages and microglia in RCS(sham) retinas, which decreased in RCS(hNPCs) retinas similar to the patterns detected in LE(sham). CONCLUSIONS: The results from this study provide evidence of the gene expression changes that occur following treatment with hNPCs in the degenerating retina. This information can be used in future studies to potentially enhance or predict responses to hNPC and other stem cell therapies for retinal degenerative diseases.

Human iPSC-Derived Neural Progenitors Preserve Vision in an AMD-Like Model.

Pluripotent stem cell-derived retinal pigment epithelial (RPE) cells are currently being tested for cell replacement in late-stage age-related macular degeneration (AMD). However, preserving vision at early-stages may also be possible. Here, we demonstrate that transplantation of neural progenitor cells (NPCs) derived from induced pluripotent stem cells (iNPCs) limits disease progression in the Royal College of Surgeons rat, a preclinical model of AMD. Grafted-iNPCs survived, remained undifferentiated, and distributed extensively in a laminar fashion in the subretinal space. Retinal pathology resulting from the accumulation of undigested photoreceptor outer segments (POS) was significantly reduced in iNPC-injected rats compared with controls. Phagosomes within grafted-iNPCs contained POS, suggesting that iNPCs had compensated for defective POS phagocytosis by host-RPE. The iNPC-treated eyes contained six to eight rows of photoreceptor nuclei that spanned up to 5 mm in length in transverse retinal sections, compared with only one row of photoreceptors in controls. iNPC treatment fully preserved visual acuity measured by optokinetic response. Electrophysiological recordings revealed that retina with the best iNPC-protected areas were 140-fold more sensitive to light stimulation than equivalent areas of contralateral eyes. The results described here support the therapeutic utility of iNPCs as autologous grafts for early-stage of AMD.

Effects of L-carnitine on follicular survival and graft function following autotransplantation of cryopreserved-thawed ovarian tissues.

The aim of this study was to investigate the effects of L-carnitine (LC) on follicular survival and ovarian function following cryopreservation-thawing and autotransplantation of ovarian tissues. ICR mice were divided into three groups: control; saline group (cryopreservation+autograft+saline); and LC group (cryopreservation+autograft+L-carnitine). The ovarian tissues from control group, saline group, and LC group were histological assessed. There were no significant differences in the percentage of morphologically normal primordial follicles between the LC group and the saline group. After 28 days of autotransplantation, apoptosis rates, plasma malondialdehyde (MDA), progesterone (P4) and estradiol (E2) concentrations, and follicular densities of grafts were evaluated. Apoptosis rate and the concentration of MDA in the LC group were significantly lower than those in the saline group. The concentration of E2 and follicular densities of grafts in LC group were significantly higher than that in saline group. LC inhibits follicle apoptosis and increases follicular survival and function of ovarian graft.

Antitumor effect and biological pathways of a recombinant adeno-associated virus as a human renal cell carcinoma suppressor.

The aims of this work are to study the antitumor effect of the adeno-associated virus on the xenografted tumors of chick embryo chorioallantoic membrane and predict potential genes and biological pathways which are associated with renal cell carcinoma. The adeno-associated virus NT4-TAT-6 × His-VHLbeta was constructed and identified. Then, chick embryos with xenografted tumor were divided into three groups and respectively inoculated with rAAV/NT4-TAT-6 × His-VHLbeta (group A), empty virus (group B), and phosphate-buffered saline (group C, the control subject). Antitumor effect in each group was investigated by means of immunofluorescence observation. Genes interacted with von Hippel-Lindau were screened by Search Tool for the Retrieval of Interacting Genes/Proteins database, while pathway analysis were performed based on Kyoto Encyclopedia of Genes and Genomes. The growth of xenografted tumors inoculated with recombinant adeno-associated virus was slower than the control subjects. The tumor volumes of group A showed significant difference compared with group B and group C (P < 0.05). Growth of xenografted tumors which administered with the recombinant adeno-associated virus was inhibited. Among the protein-protein interaction network, TCEB2, HIF1A, TCEB1, CUL2, RBX1, and PHF17 were hub genes which might be involved in the development of renal cell carcinoma. The most significant signaling pathway was renal cell carcinoma. In this paper, we constructed and identified the recombinant adeno-associated virus NT4-TAT-6 × His-VHLbeta and studied the antitumor effect of the adeno-associated virus on xenografted tumors of chicken embryo chorioallantoic membrane. In addition, genes in the protein-protein interaction network which are associated with renal cell carcinoma were revealed and the biological pathway of renal cell carcinoma was identified. Our results provide a gene-therapeutic agent for the treatment of human renal cell carcinoma.

Design and Implementation of Electrochromic Smart Windows with Self-Driven Thermoelectric Power Generation.

Electrochromic smart windows can achieve controllable modulation of color and transmittance under an external electric field with active light and thermal control capabilities, which helps reduce energy consumption caused by building cooling and heating. However, electrochromic smart windows often rely on external power circuits, which greatly affects the independence and portability of smart windows. Based on this, an electrochromic smart window driven by temperature-difference power generation was designed and implemented. This smart window provides automatic and manual control of the reversible cycle of electrochromic glass from light blue to dark blue according to user requirements and changes in the surrounding environment, achieving adaptive adjustment of visual comfort and reducing energy consumption. The infrared radiation rejection (from 780 to 2500 nm) of the electrochromic smart window is as high as 77.3%, and its transmittance (from 380 to 780 nm) fluctuates between 39.2% and 56.4% with changes in working state. Furthermore, the temperature in the indoor simulation device with electrochromic glass as the window was 15 °C lower than that with ordinary glass as the window after heating with a 250 W Philips infrared lamp for ten minutes. After 2000 cycles of testing, the performance of the smart window was basically maintained at its initial values, and it has broad application prospects in buildings, vehicles, and high-speed rail systems.

Retinal morphological and functional changes in an animal model of retinitis pigmentosa.

The P23H-1 transgenic rat carries a mutated mouse opsin gene, in addition to endogenous opsin genes, and undergoes progressive photoreceptor loss that is generally characteristic of human autosomal dominant retinitis pigmentosa (RP). Here, we examined morphological changes correlated with visual function that is comparable to clinical application in the pigmented P23H-1 rat retina as photoreceptor degeneration progressed. We found that rod function was compromised as early as postnatal day 28 and was a good indicator for tracking retinal degeneration. Cone function was normal and did not change until the thickness of the photoreceptor layer was reduced by 75%. Similar to the threshold versus intensity curves used to evaluate vision of RP patients, light-adaptation curves showed that cone thresholds depended on the number of remaining functioning cones, but not on its length of outer segments (OS). By 1 year of age, both rod and cone functions were significantly compromised. Correlating with early abnormal rod function, rods and related secondary neurons also underwent progressive degeneration, including shortening of inner and OS of photoreceptors, loss of rod bipolar and horizontal cell dendrites, thickening of the outer Müller cell processes, and reduced density of pre- and postsynaptic markers. Similar early morphological modifications were also observed in cones and their related secondary neurons. However, cone function was maintained at nearly normal level for a long period. The dramatic loss of rods at late stage of degeneration may contribute to the dysfunction of cones. Attention has to be focused on preserving cone function and identifying factors that damage cones when therapeutic regimes are applied to treat retinal degeneration. As such, these findings provide a foundation for future studies involving treatments to counter photoreceptor loss.

MFN1 augmentation prevents retinal degeneration in a Charcot-Marie-Tooth type 2A mouse model.

Charcot-Marie-Tooth disease type 2A (CMT2A), the most common inherited peripheral axonal neuropathy, is associated with more than 100 dominant mutations, including R94Q as the most abundant mutation in the Mitofusin2 (MFN2) gene. CMT2A is characterized by progressive motor and sensory loss, color-vision defects, and progressive loss of visual acuity. We used a well-established transgenic mouse model of CMT2A with R94Q mutation on MFN2 gene (MFN2 R94Q ) to investigate the functional and morphological changes in retina. We documented extensive vision loss due to photoreceptor degeneration, retinal ganglion cell and their axonal loss, retinal secondary neuronal and synaptic alternation, and Müller cell gliosis in the retina of MFN2 R94Q mice. Imbalanced MFN1/MFN2 ratio and dysregulated mitochondrial fusion/fission result in retinal degeneration via P62/LC3B-mediated mitophagy/autophagy in MFN2 R94Q mice. Finally, transgenic MFN1 augmentation (MFN2 R94Q :MFN1) rescued vision and retinal morphology to wild-type level via restoring homeostasis in mitochondrial MFN1/MFN2 ratio, fusion/fission cycle, and PINK1-dependent, Parkin-independent mitophagy.

Differential Susceptibility of Fetal Retinal Pigment Epithelial Cells, hiPSC- Retinal Stem Cells, and Retinal Organoids to Zika Virus Infection.

Zika virus (ZIKV) causes microcephaly and congenital eye disease. The cellular and molecular basis of congenital ZIKV infection are not well understood. Here, we utilized a biologically relevant cell-based system of human fetal retinal pigment epithelial cells (FRPEs), hiPSC-derived retinal stem cells (iRSCs), and retinal organoids to investigate ZIKV-mediated ocular cell injury processes. Our data show that FRPEs were highly susceptible to ZIKV infection exhibiting increased apoptosis, whereas iRSCs showed reduced susceptibility. Detailed transcriptomics and proteomics analyses of infected FRPEs were performed. Nucleoside analogue drug treatment inhibited ZIKV replication. Retinal organoids were susceptible to ZIKV infection. The Asian genotype ZIKV exhibited higher infectivity, induced profound inflammatory response, and dysregulated transcription factors involved in retinal organoid differentiation. Collectively, our study shows that ZIKV affects ocular cells at different developmental stages resulting in cellular injury and death, further providing molecular insight into the pathogenesis of congenital eye disease.

Human Neural Progenitors Expressing GDNF Enhance Retinal Protection in a Rodent Model of Retinal Degeneration.

Stem cell therapy for retinal degenerative diseases has been extensively tested in preclinical and clinical studies. However, preclinical studies performed in animal models at the early stage of disease do not optimally translate to patients that present to the clinic at a later stage of disease. As the retina degenerates, inflammation and oxidative stress increase and trophic factor support declines. Testing stem cell therapies in animal models at a clinically relevant stage is critical for translation to the clinic. Human neural progenitor cells (hNPC) and hNPC engineered to stably express GDNF (hNPCGDNF) were subretinally injected into the Royal College of Surgeon (RCS) rats, a well-established model for retinal degeneration, at early and later stages of the disease. hNPCGDNF treatment at the early stage of retinal degeneration provided enhanced visual function compared to hNPC alone. Treatment with both cell types resulted in preserved retinal morphology compared to controls. hNPCGDNF treatment led to significantly broader photoreceptor protection than hNPC treatment at both early and later times of intervention. The phagocytic role of hNPC appears to support RPE cell functions and the secreted GDNF offers neuroprotection and enables the extended survival of photoreceptor cells in transplanted animal eyes. Donor cells in the RCS rat retina survived with only limited proliferation, and hNPCGDNF produced GDNF in vivo. Cell treatment led to significant changes in various pathways related to cell survival, antioxidative stress, phagocytosis, and autophagy. A combined stem cell and trophic factor therapy holds great promise for treating retinal degenerative diseases including retinitis pigmentosa and age-related macular degeneration.